CIC-DUX4 oncoprotein drives sarcoma metastasis and tumorigenesis via distinct regulatory programs

Ross A. Okimoto, … , Tadashi Kondo, Trever G. Bivona

J Clin Invest. 2019;129(8):3401-3406. https://doi.org/10.1172/JCI126366.

Concise Communication Oncology

Transcription factor fusion create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua–double 4 (CIC-DUX4) is a factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4– expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4– expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications.

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CIC-DUX4 oncoprotein drives sarcoma metastasis and tumorigenesis via distinct regulatory programs

Ross A. Okimoto,1,2 Wei Wu,1 Shigeki Nanjo,1 Victor Olivas,1 Yone K. Lin,1 Rovingaile Kriska Ponce,1 Rieko Oyama,3 Tadashi Kondo,3 and Trever G. Bivona1,2,4 1Department of Medicine, 2Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA. 3Division of Rare Cancer Research, National Cancer Center Research Institute, Tokyo, Japan. 4Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.

Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua–double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4–expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4–expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications.

Introduction functions as a transcriptional activator instead of a transcriptional The biological regulation of transcription factors and repressor pro- repressor (5, 6). This property suggests that the C-terminal DUX4 teins is an essential mechanism for maintaining cellular homeo- binding partner may confer neomorphic transcriptional regulatory stasis, and is often dysregulated in human cancer (1). Indeed, properties to CIC, while retaining WT CIC DNA binding specificity. chromosomal rearrangements involving transcriptional regula- ETV4 is one of the most well-characterized transcriptional targets tory genes that lead to transcriptional dysregulation are present of CIC-DUX4 (5, 7, 8). More than 90% of CIC-DUX4–expressing in many cancers, including approximately 30% of all soft tissue tumors show ETV4 upregulation by IHC, which distinguishes them sarcomas (2). The majority of these oncogenic fusions involve from other small round blue cell sarcomas (8). We and others have transcription factors or regulators that are not readily “drugga- demonstrated a prometastatic function for ETV4 overexpression in ble” in a direct pharmacologic manner and thus have proven dif- tumors with inactivation of WT CIC (9, 10). The functional role of ficult to therapeutically target in the clinic. A prime example is the ETV4 in CIC-DUX–positive tumors is unknown. CIC-DUX4 fusion oncoprotein, which fuses capicua (CIC) to the Beyond ETV4, the identity and function of other CIC target genes double homeobox 4 , DUX4. CIC-DUX4–positive soft tissue are less well defined. Intriguingly, recent studies showed increased tumors are an aggressive subset of undifferentiated round cell sar- expression of cell-cycle-regulatory genes in CIC-rearranged sarco- comas that arise in children and young adults. Despite histological mas, although the functional relevance of this observation for onco- similarities to Ewing sarcoma, CIC-DUX4–positive sarcomas are genesis and cancer growth in this context is unclear (6, 7). clinically distinct and typically characterized by the rapid devel- Here, we developed a range of in vitro and in vivo cancer opment of lethal metastatic disease and chemoresistance (3). By model systems to define the mechanism by which CIC-DUX4 revealing downstream molecular targets that relay the functional controls capicua-regulated transcriptional pathways to promote output of CIC-DUX4, we can potentially identify and develop a hallmark features of malignancy, including tumor cell survival, more rational therapeutic strategy to improve patient outcomes. growth, and metastasis. CIC is a transcriptional repressor (4). The CIC-DUX4 fusion structurally retains greater than 90% of native CIC, yet it Results and Discussion We recently reported that inactivation of native CIC derepresses an ETV4-MMP24–mediated prometastatic circuit (9). Since ETV4 Conflict of interest: TGB is an advisor to Revolution Medicines, Novartis, AstraZen- is a direct transcriptional target of CIC-DUX4 (Supplemental Fig- eca, Takeda, SpringWorks, Jazz, and Array BioPharma and receives research funding ure 1A; supplemental material available online with this article; from Revolution Medicines and Novartis. https://doi.org/10.1172/JCI126366DS1) (6, 7), we hypothesized Copyright: © 2019, American Society for Clinical Investigation. that the high metastatic rate observed in patients harboring CIC- Submitted: November 21, 2018; Accepted: May 24, 2019; Published: July 22, 2019. Reference information: J Clin Invest. 2019;129(8):3401–3406. DUX4 fusions was dependent on ETV4 expression. To explore this https://doi.org/10.1172/JCI126366. hypothesis, we developed an orthotopic soft tissue metastasis mod-

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Figure 1. ETV4 promotes metastasis in CIC-DUX4–expressing sarcoma. (A) Schematic of the orthotopic soft tissue metastasis model. (B) Representative bioluminescence (BLI) images from mice bearing CIC-DUX4–expressing NIH 3T3 cells with shCtrl (n = 9) or shETV4 (n = 7). (C) Immunoblot of ETV4 and MMP24 from NIH 3T3 cells expressing empty vector (EV), shCtrl, shETV4a, or shETV4b. (D) Number of lung metastases in mice bearing CIC-DUX4–express- ing NIH 3T3 cells with shCtrl (n = 9) or shETV4 (n = 7). P value, Student’s t test. (E) Transwell invasion assay comparing CIC-DUX4–expressing NIH 3T3 cells with EV, shCtrl, shETV4a, or shETV4b. P value, 1-way ANOVA. Error bars represent SEM. (F) Relative photon flux from mice orthotopically implanted with CIC-DUX4–expressing NIH 3T3 cells expressing either shCtrl or shETV4. Error bars reflect SEM. (G) Subcutaneously implanted NIH 3T3 cells with EV, CIC- DUX4, or CIC-DUX4 plus shETV4a or shETVb. P value, 1-way ANOVA. Error bars represent SEM.

el utilizing luciferase-based imaging to track tumor dissemination ed that the CIC-DUX4 fusion can regulate cell cycle gene expres- in vivo (Figure 1A). This system produces rapid pulmonary metas- sion (6, 7). We established that ectopic expression of CIC-DUX4 tases, accurately recapitulating metastatic tumor dissemination increased NIH 3T3 cell growth in vitro and in vivo through enhanced in patients (3). Using this in vivo system, we engineered NIH 3T3 cell cycle progression (Supplemental Figure 2, A–E). The CIC-DUX4 mouse fibroblasts, which offer the advantage of a genetically con- fusion increased the number of cells progressing through S-phase, as trolled system (as with the study of other oncoproteins), to express reflected by an increased G2/M fraction compared with control (Sup- the CIC-DUX4 fusion oncoprotein (11–13). We observed rapid pri- plemental Figure 2F). In order to identify CIC-DUX4–regulated cell mary tumor formation at the site of implantation in 100% of the cycle genes, we leveraged a publicly available microarray-based data injected mice (Figure 1B). Mice with genetic silencing of ETV4 set (GSE60740) to perform a comparative transcriptional analysis showed decreased expression of its established target, MMP24 between CIC-DUX4–replete (IB120 EV cell line) and 2 independent (9) (Figure 1C, and Supplemental Figure 1, B and C), and impaired CIC-DUX4–knockdown (KD) patient-derived cell lines (IB120 shCIC- metastatic efficiency in vivo and invasive capacity in vitro com- DUX4a and IB120 shCIC-DUX4b) (14). Notably, in the context of the pared with mice bearing a control silencing vector (Figure 1, B–E). CIC-DUX4 fusion, CIC retains it binding specificity but acquires acti- While ETV4 suppression decreased pulmonary metastases, it did vating properties (5). Hence, we focused our attention on genes that not have a profound impact on tumor growth when CIC-DUX4– were downregulated upon CIC-DUX4 KD. We observed 165 shared expressing cells were implanted either orthotopically or subcuta- downregulated (and 105 upregulated) genes between the 2 indepen- neously into the flank of immunocompromised mice (Figure 1, F dent shCIC-DUX4 data sets (IB120 shCIC-DUX4a and IB120 shCIC- and G). These findings suggest that the primary function of CIC- DUX4b) (Figure 2A, and Supplemental Tables 1 and 2). Functional DUX4–mediated ETV4 upregulation is to promote invasion and clustering of the 165 putative CIC-DUX4 target genes revealed enrich- metastasis, but not tumor cell proliferation or tumor growth per se. ment for genes that regulate DNA replication and cell cycle machinery We next investigated transcriptional targets and programs that (Figure 2B) (15). We compared the expression of these 165 genes in 14 could regulate other key aspects of tumor biology beyond metastasis, CIC-DUX4 and 7 EWS-NFATC2 patient-derived tumors that were including tumor cell proliferation and growth. Prior studies suggest- profiled on the same Affymetrix array (GSE60740). We observed a

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Figure 2. CIC-DUX4 regulates cell cycle genes. (A) Schematic algorithm to identify putative CIC-DUX4 target genes. (B) Functional annotation of CIC- DUX4–activated genes in IB120 cells. (C) Heatmap depicting the 165 CIC-DUX4–activated genes identified in A across 14 CIC-DUX4 and 7 EWSR1-NFATc2 patient-derived tumors. Cell cycle gene symbols are magnified.

significant increase in the expression of multiple cell-cycle-regulatory ty vector control (Figure 3D). These data show that CCNE1 is a direct genes in CIC-DUX4–expressing tumors (Figure 2C). Our findings transcriptional target that is upregulated by CIC-DUX4. extend recent studies (6, 7) and indicate that targeting the cell cycle To explore the functional role of CCNE1 in CIC-DUX4–express- in CIC-DUX4–expressing tumors is a potential therapeutic approach. ing tumors, we genetically silenced CCNE1 in CIC-DUX4–express- In order to identify direct transcriptional targets of the CIC- ing NIH 3T3 cells (a genetically controlled system) and observed DUX4 fusion, we first surveyed the promoters (within –2 kb and +150 decreased growth in vitro and in vivo compared with control (Fig- bp of the transcription start site) of all 165 putative response genes ure 3, E–G, and Supplemental Figure 3, A–C). While there is no val- for the highly conserved CIC-binding motif (T[G/C]AATG[A/G]A) idated pharmacologic strategy to directly block CCNE1, inhibition (5). Using this systematic approach, we identified 43 genes, includ- of the CCNE1 binding partner CDK2 has therapeutic efficacy in ing the known CIC target genes ETV1, -4, and -5 and multiple regu- other cancer types (16–18). We hypothesized that inhibiting CDK2 lators of the cell cycle (Supplemental Table 3). Since ectopic expres- in CIC-DUX4–expressing cells with the established small mole- sion of CIC-DUX4 promoted S-phase progression (Supplemental cule drug dinaciclib (16) could limit tumor growth. To explore this Figure 2, E and F), we focused on genes that directly regulate the hypothesis, we implanted CIC-DUX4–expressing NIH 3T3 cells

G1/S transition. Of these genes, cyclin E1 (CCNE1) expression was subcutaneously into immunodeficient mice and treated them with consistently upregulated in CIC-DUX4–expressing tumors (Figure low-dose dinaciclib (20 mg/kg/d); we observed decreased growth 2C). We therefore investigated whether CIC-DUX4 transcription- compared with vehicle control (Figure 4, A–C) (16). These findings ally controls CCNE1 expression. To explore this hypothesis, we first suggest that pharmacologic inhibition of the CCNE-CDK2 complex localized 2 tandem CIC-binding motifs within 1 kb of the CCNE1 is a potential therapeutic strategy in CIC-DUX4–expressing tumors. transcriptional start site (Figure 3A). We next performed ChIP-PCR There are few patient-derived models of CIC-DUX4–positive analysis, which revealed CCNE1 occupancy by the CIC- tumors. Nevertheless, in order to increase the clinical relevance of DUX4 fusion (Figure 3, B and C). Additionally, a luciferase-based our findings we obtained rare, established patient-derived CIC- reporter assay demonstrated enhanced promoter activity by the DUX4–expressing cells (NCC_CDS1_X1 and NCC_CDS1_X3) (19) ectopic expression of CIC-DUX4 compared with CIC WT and emp- to test the functional impact of ETV4 KD and CDK2 inhibition.

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Figure 3. CCNE1 is a molecular target of CIC-DUX4. (A) Schematic of 2 CIC-binding motifs in the CCNE1 pro- moter. (B and C) ChIP-PCR from H1975 M1 (CIC null) cells reconstituted with CIC-DUX4, showing CIC-DUX4 occu- pancy on the CCNE1 promoter. P value, Student’s t test. Error bars represent SEM. Performed in triplicate. (D) CCNE1 luciferase promoter assay in 293T cells comparing EV with CIC WT or CIC-DUX4. P value, 1-way ANOVA. Error bars represent SEM. Performed in trip- licate. (E) Subcutaneously implanted NIH 3T3 cells expressing EV, CIC-DUX4 (n = 7), or CIC-DUX4 plus shCCNE1a (n = 7) or shCCNE1b (n = 7). P value, 1-way ANOVA. Error bars represent SEM. (F) Tumor explants from mice in E. (G) Tumor weights from mice in E. P values, 1-way ANOVA.

Ewing sarcoma), in vitro or in vivo (Supplemental Figure 6, A and B). Consistent with these findings, we observed low levels of CCNE1 in PAX3-FOXO1–positive RH30 rhab- domyosarcoma cells compared with CIC-DUX4–expressing NCC_ CDS1_X1 cells (Supplemental Fig- ure 6C). These findings further sug- We found that genetic silencing of ETV4, but not ETV1 or ETV5 gest that CIC-DUX4–expressing tumors are molecularly dependent (known CIC-DUX4 target genes) (5), decreased invasiveness but on the CCNE-CDK2 complex, which is a potential therapeutic target. did not impact tumor growth or apoptosis (Supplemental Figure To further demonstrate the therapeutic specificity of targeting 4, A–G). Additionally, we found that CIC-DUX4–expressing cells CDK2, we used the clinical CDK4/6 inhibitor palbociclib, which has were exquisitely sensitive to nanomolar (nM) concentrations no substantial activity against CDK2, and found no impact on tumor of 2 established, independent CDK2 inhibitors, dinaciclib and growth or apoptosis in NCC_CDS1_X1 cells (Supplemental Figure SNS-032 (16, 20) (Figure 4D and Supplemental Figure 5A). The 7A). Moreover, while genetic silencing of CDK2 with siRNAs did not effects on tumor viability were largely mediated through apoptotic impact cell invasion, it did decrease cell number and viability in CIC- cell death as measured by poly(ADP-ribose) polymerase (PARP) DUX4–expressing NCC_CDS1_X1 cells compared with control (Sup- cleavage and caspase activity, again indicating that CIC-DUX4– plemental Figure 7, B–E). While we did not observe an effect on viabil- expressing tumors are dependent on the CCNE-CDK2 complex ity with CCNE1 KD alone, combined CCNE1 and CCNE2 KD reduced for survival (Figure 4, E–G, and Supplemental Figure 5, B and C). viability to similar levels as siCDK2 cells (Supplemental Figure 7, C, To mitigate the off-target effects of dinaciclib and SNS-032, we D, F, and G). These findings are consistent with prior observations performed genetic silencing of CDK7 and CDK9, which did not that both CCNE1 and CCNE2 converge on and activate CDK2, which suppress NCC_CDS1_X1 growth (Supplemental Figure 5, D–G). our pharmacologic and genetic studies described above indicate is We next tested whether CDK2 inhibition could specifically sup- required for CIC-DUX4–expressing tumor survival (21). press the growth of patient-derived CIC-DUX4–expressing cells in Our data suggest that in human-derived NCC_CDS1_X1 cells, vivo. To address this hypothesis, we generated subcutaneous tumor CCNE2 can compensate for CCNE1 loss. Consistent with this notion, xenografts of the patient-derived CIC-DUX4–expressing cells in we observed approximately 100-fold upregulation of CCNE2 mRNA immunodeficient mice and found that dinaciclib limited tumor upon genetic silencing of CCNE1 and through ChIP-Seq analysis growth compared with vehicle control treatment (Figure 4, H–J). The identified CIC-DUX4 binding to the promoter of CCNE1 but not decreased tumor growth observed in dinaciclib-treated mice was CCNE2 (Supplemental Figure 8, A–C) in human NCC_CDS1_X1 accompanied by increased PARP cleavage in tumor explants, consis- cells. In contrast to these findings, we did not observe a compensa- tent with the apoptotic effect observed in vitro (Figure 4K). The impact tory increase in CCNE2 mRNA with CCNE1 KD in NIH 3T3 (mouse) on tumor growth following CDK2 inhibition was relatively specific for cells expressing CIC-DUX4 (Supplemental Figure 8, D and E). The CIC-DUX4–expressing tumors, as we did not observe a significant lack of CCNE2 upregulation in CIC-DUX4–expressing NIH 3T3 cells response to dinaciclib in other sarcoma subtypes that harbor distinct is a plausible explanation for the growth-suppressive effect of CCNE1 transcription factor fusion oncoproteins (rhabdomyosarcoma or KD in our initial NIH 3T3 system (Figure 3E), a phenotype that was

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Figure 4. The CCNE-CDK2 complex is a therapeutic target in CIC-DUX4 sarcoma. (A) Subcutaneously implanted NIH 3T3 cells expressing CIC-DUX4 and treated with vehicle (n = 8) or dinaciclib (n = 8). P value, Student’s t test. Error bars represent SEM. (B) Tumor explants from mice in A. (C) Tumor weights of mice in A. P value, Student’s t test. (D) Patient-derived CIC-DUX4–expressing cells (NCC_CDS1_X1 and NCC_CDS1_X3) treated with dinaciclib or DMSO. Performed in duplicate. (E) Immuno- blot of phosphorylated retinoblastoma (RB), PARP, and actin control in NCC_CDS1_ X3 cells treated with dinaciclib. Representative figure; performed in duplicate. (F) Relative caspase-3/7 activity in NCC_CDS1_X1 and (G) NCC_CDS1_X3 cells treated with dinaciclib or DMSO. *P = 0.01, **P < 0.0001, 1-way ANOVA. Error bars represent SEM. (H) Subcutaneously implanted NCC_CDS1_X3 cells treated with vehicle control (n = 6) or dinaciclib (n = 7). P value, Student’s t test. Error bars reflect SEM. (I) Tumor weights from mice treated in H. P value, Student’s t test. (J) Tumor explants from mice in H. (K) Immunoblot of phosphorylated RB, total and cleaved PARP, and actin control from a NCC_CDS1_X3 tumor explant treated with vehicle (Veh) or dinaciclib. Representative figure; performed in duplicate.

not shared with human-­derived NCC_CDS1 cells (Supplemental Fig- fusion oncoprotein that promote either tumor growth or metastasis. ure 7, C and D). Importantly, we identified CIC-DUX4–binding sites We reveal that ETV4 is a conserved target gene that enhances the in the promoter of mouse CCNE1, but not CCNE2 (–4 kb to +100 of metastatic capacity of CIC-DUX4–expressing tumors, without sig- the transcription start site), suggesting that CCNE1 (but not CCNE2) nificantly impacting tumor growth. These findings extend previous is a conserved transcriptional target in human (NCC_CDS1) and data that demonstrate a prometastatic role for ETV4 in certain human mouse (NIH 3T3 expressing CIC-DUX4) cells (Supplemental Tables cancers (9, 22). Targeting an ETV4-mediated transcriptional program 3 and 4, and Supplemental Figure 8F). Our mechanistic dissection of downstream of CIC-DUX4 can potentially limit tumor dissemination. CIC-DUX4–expressing tumors reveals a unique molecular and ther- Further analysis of CIC-DUX4–bearing tumors coupled with apeutic dependence on the CCNE-CDK2 cell cycle complex. Our our functional studies also identified a dependence on specific cell data show that this dependence can be exploited with clinical CDK2 cycle machinery to enhance tumor growth but not invasive capaci- inhibitors that limit tumor growth through apoptotic induction in a ty. We reveal that the CCNE-CDK2 complex is a molecular target of tumor type with few effective therapies. the CIC-DUX4 oncoprotein that controls tumor growth and survival. The CIC-DUX4 fusion oncoprotein is a relatively understudied While others have observed transcriptional upregulation of cell cycle molecular entity that characterizes a rare but lethal subset of undiffer- genes in CIC-DUX4–expressing tumors (6, 7), our data establish the entiated round cell sarcomas. We undertook a mechanistic dissection CCNE-CDK2 complex as a direct molecular target that can be phar- of the molecular function of the CIC-DUX4 fusion oncoprotein and macologically exploited with clinically developed CDK2 inhibitors. report unique dependencies on specific transcriptional targets of the The therapeutic impact may extend beyond the CIC-DUX4 fusion

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to include other CIC-fused oncoproteins. Recent findings reveal Statistics. Experimental data are presented as mean ± SEM. P values a shared transcriptional program downstream of all known CIC derived for all in vitro experiments were calculated using 2-tailed Student’s fusions, including CIC-FOXO4 and CIC-NUTM1 (23). These find- t test or 1-way ANOVA. P < 0.05 was considered statistically significant. ings suggest that many CIC-fused tumors retain their CIC-binding Study approval. For tumor xenograft studies, including orthotopic specificity while converting native CIC into a transcriptional acti- and subcutaneous models, specific pathogen–free conditions and vator instead of a repressor in a neomorphic manner. It would be facilities were approved by the American Association for Accredita- compelling to explore whether the CCNE-CDK2 complex or other tion of Laboratory Animal Care. Surgical procedures were reviewed components of the cell cycle machinery drive tumor growth in these and approved by the UCSF IACUC, protocol AN107889-03A. other CIC-fused tumor types, an area for future investigation. Our mechanistic dissection of the downstream molecular targets Author contributions of CIC-DUX4 provides therapeutically relevant insight for target- RAO designed and performed the experiments, analyzed the ing ETV4-mediated metastasis and CCNE-CDK2–regulated tumor data, and wrote the manuscript. RAO, VO, SN, YKL, RKP, RO, growth. Our data support a broader conceptual paradigm in which and TK performed in vitro and in vivo mouse studies. WW per- certain transcription factor fusion oncoproteins, such as CIC-DUX4, formed bioinformatics. TK performed experiments and provided utilize neomorphic and distinct downstream regulatory programs to cell lines. TGB directed the project, analyzed experiments, and control divergent cancer hallmarks, such as proliferative capacity and wrote the manuscript. metastatic competency. The data offer a potential mechanistic expla- nation for the pleiotropic functions of this important class of oncopro- Acknowledgments teins (i.e., transcription factor fusion oncoproteins such as CIC-DUX4). RAO was supported by a grant from the A.P. Giannini Foun- Our findings highlight the clinical importance of molecular sub- dation and NIH 1K08CA222625-01. TGB acknowledges sup- classification of morphologically similar tumor types, such as small port from NIH/NCI U01CA217882, NIH/NCI U54CA224081, round cell sarcomas. Identifying the different fusion oncoproteins NIH/NCI R01CA204302, NIH/NCI R01CA211052, NIH/NCI present in clinical samples paves the way for oncoprotein fusion–spe- R01CA169338, and the Pew and Stewart Foundations. cific therapeutic targeting to improve patient outcomes. Our study highlights the utility of elucidating the mechanistic features of tumors Address correspondence to: Ross A. Okimoto, 513 Parnas- that are driven by transcription factor fusion oncoproteins to identify sus Avenue, HSW1201, San Francisco, California 94143, USA. precision medicine–based molecular therapeutic strategies. Phone: 415.514.1083; Email: [email protected]. Or to: Trever G. Bivona, 600 16th Street, Genentech Hall, N232, San Methods Francisco, California 94158, USA. Phone: 415.502.0237; Email: Refer to Supplemental Methods for details. [email protected].

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